Test apparatus for assaying a component in a liquid sample

ABSTRACT

A test apparatus for assaying a component in a liquid sample by measuring a reflected light comprising a support having a through hole or a light permeable area, a reagent layer having a detecting area fixed on the support to cover said through hole or light permeable area, and a cover which covers at least the detecting area, wherein at least a portion covering the detecting area in the cover is processed into a color which substantially has no influence on the reflected light at a measured wavelength.

FIELD OF THE INVENTION

[0001] The present invention relates to a test apparatus which are usedfor assaying a specific component contained in a liquid sample,especially a blood sample such as whole blood and serum, and a bodyfluid such as urine and cerebrospinal fluid by a color reaction. Moreparticularly, the present invention relates to a test apparatus usingreflected light as a measuring means.

BACKGROUND OF THE INVENTION

[0002] In order to measure a specific component in a liquid samplequickly and simply, for example, glucose, cholesterol or the like in ablood sample, or glucose, hemoglobin or the like in a body fluid sample,a dry type test apparatus comprising a support having provided thereon areagent layer containing at least a reagent capable of developing acolor by a reaction with the specific component.

[0003] When the test apparatus is used, the reaction starts by thesupply of a liquid sample to one side of the reagent layer.

[0004] A color density of the developed color on the reagent layer afterthe reaction with a specific component corresponds to the amount of thespecific component in the liquid sample. Accordingly, the specificcomponent in the liquid sample can be determined by measuring the colordensity. The color density of the developed color on the reagent layeris measured by applying an incident light to the reagent layer after thecolor reaction and detecting intensity of the resulting reflected lightor transmitted light.

[0005] The test apparatuses using the reflected light can be dividedroughly into two types from the viewpoint of sample supply to thereagent layer; one is a test apparatus having a sample-supplying areaand a detecting area (namely, an incident light side area) on the sameside, and another is a test apparatus having a sample-supplying area anda detecting area on different sides.

[0006] The test apparatus having a sample-supplying area and a detectingarea on the same side is useful in measuring samples having hightransparency such as sera and urine, but not suitable for measuringhaving low transparency such as whole blood.

[0007] On the other hand, the test apparatus having a sample-supplyingarea and a detecting area on different sides is markedly advantageous,because it is fully possible to measure a sample having low transparencysuch as whole blood by forming a reflection layer or a separation layeron the reagent layer.

[0008] In the test apparatus having a sample-supplying area and adetecting area on different sides, however, not only the reagent layerbut also the structure of the test apparatus itself become complex incomparison with the test apparatus having a sample-supplying area and adetecting area on the same side. Accordingly, new components are oftenrequired in addition to the reagent layer and support. Examples of thetest apparatus having a sample-supplying side and a detecting side ondifferent sides include those which are disclosed in JP-A-55-59326 andJP-A-4-188065 (the term “JP-A” as used herein means an “unexaminedpublished Japanese patent application”).

[0009] The reflection layer is provided in order to clarify a coloringdegree of the reagent layer by reflecting the injected light, and whiteparticles of titanium dioxide or the like are used as its material. Theusually used reflection layer has a thickness of from 2 μm to 50 μm.Also, light reflection capacity is added to the reagent layer itself bykneading titanium dioxide with a reagent in the reagent layer.

[0010] However, the reflection layer has a disadvantage in that itcannot perform complete reflection of light so that the light partiallypasses through the layer. When the thickness of the light reflectionlayer is increased in order to prevent such an unnecessary passage oflight, the permeability of the liquid sample becomes poor so that properresults cannot be obtained.

[0011] In the case of the test apparatus of JP-A-4-188065, the portioncorresponding to the reagent layer, as shown in FIG. 2 in expanded view,comprises a porous membrane to be used as a sample-holding layer, areagent layer prepared from a reagent, a buffer, a binder and the like,a light reflection layer prepared from titanium dioxide which also actsas a blood cell filtration layer, a space where the supplied liquidsample passes, and a cover which forms a space above the reagent layer.

[0012] When light is injected into the reagent layer from the porousmembrane side, types of the resulting reflected light are dividedroughly into (1) light reflected from the porous membrane surface, (2)light reflected from the inner portion of the porous membrane and theinner portion of the reagent layer, (3) light reflected from the lightreflection layer and (4) light reflected from the cover. Althoughnecessary information among them is only the reflected light of (2) and(3), other types of reflected light are also measured. That is, thereflected light obtained by the injection of light is a mixture ofseveral types of reflected light, and these unnecessary types ofreflected light are generally measured as an error.

[0013] Even in the case of the same type of liquid sample such as wholeblood, hemolytic serum, chromaturia or the like, the liquid sampleitself sometimes shows different colors. For example, the color of wholeblood varies depending on the difference in its hematocrit value and thelike, and the color of serum varies depending on the difference in itsdegree of hemolysis and the like. Depending on the difference in thecolor of these liquid samples, coloration of the reagent layer variesand the amount of light passing through the reagent layer also varies.

[0014] When the measurement is carried out using reflected light, thelight reflected from the cover provided on the side which is not theincident direction of the reagent layer (the aforementioned type (4)reflected light) is also simultaneously detected by its passage throughthe reagent layer, so that difference in the coloration of the reagentlayer in each measurement causes changes in the amount of light whichpasses through the reagent layer and also the amount of the reflectedlight of type (4) which passed through the same, thus inevitablyexerting unnecessary influence upon the measured values.

SUMMARY OF THE INVENTION

[0015] Therefore, an object of the present invention is to resolve theabove-described problems concerning irregular measured values due toerrors caused by the variation of color of each liquid sample in a testapparatus in which a specific component in a liquid sample is determinedby measuring a reflected light.

[0016] In order to resolve the above problems, the inventors of thepresent invention have conducted intensive studies and found that theinfluence of unnecessary reflection light can be avoided when an areawhich substantially having no influence on the reflected light at ameasured wavelength is provided on one side of a reagent layer oppositeto the side of light incident direction, because the reflection of lightpassed through a reflection layer in the reagent layer is lowered in thearea.

[0017] Accordingly, this and other objects of the present invention havebeen attained by a test apparatus for assaying a component in a liquidsample by measuring a reflected light comprising

[0018] a support having a through hole or a light permeable area,

[0019] a reagent layer having a detecting area fixed on the support tocover the through hole or light permeable area, and

[0020] a cover which covers at least the detecting area,

[0021] wherein at least a portion covering the detecting area in saidcover is processed into a color which substantially has no influence onsaid reflected light at a measured wavelength.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a sectional view of a test apparatus according to thepresent invention.

[0023]FIG. 2 is an expanded sectional view of the reagent layer and itssurrounding area shown in FIG. 1.

[0024]FIG. 3 is a sectional view of another example of test apparatusaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The test apparatus for use in the present invention can have thefollowing embodiments. One of them is a test apparatus having the samebasic structure disclosed in JP-A-4-188065, in which its cover is fixedon a support in such a manner that a sample-holding room is formedbetween the reagent layer and the cover which has a sample-supplyinghole and an air exhaust hole. This cover is processed into a color whichsubstantially has no influence on the reflected light at a measuredwavelength (see sectional views of FIGS. 1 and 2, FIG. 2 is an expandedview of the reagent layer and its surrounding area of FIG. 1).

[0026] When this test apparatus is used, a liquid sample is addeddropwise through the sample-supplying hole. The thus added liquid sampledevelops in the sample-holding room and reaches the reagent layer set ata halfway position of the sample-holding room. When the sample is wholeblood, blood plasma alone is separated by the blood cell separatingaction in the reagent layer, and reacts with the reagent in a porousmembrane which acts as a sample-holding layer.

[0027] The measurement is carried out by observing the porous membranefrom the support side.

[0028] In another embodiment of the test apparatus, a cover is directlycontacted with one side of a reagent layer opposite to the support (seeFIG. 3). That is, the reagent layer is fixed on the support, and aportion of the reagent layer covered with the cover corresponds to thedetecting area.

[0029] When this test apparatus is used, a liquid sample is spotted onposition A of the reagent layer. Thereafter, the liquid sample developsin the reagent layer and mixed therewith, and a reaction of blood plasmawith the reagent occurs in the porous membrane which acts as asample-holding layer. That is, the porous membrane of the test apparatusalso acts as a matrix to transport the liquid sample.

[0030] According to the reagent layer of the present invention, it ispreferable to coat or impregnate a base material with a coloring reagentfor an enzyme reaction, a chemical reaction or the like. The basematerial has light permeability and is excellent in performingdevelopment and permeation of liquid. Examples thereof include matricessuch as filter paper, cloth and glass filter, and gels such as gelatin.

[0031] Examples of the coloring reagent for use in the present inventioninclude 4-aminoantipyrine, Trinder's reagents (e.g., DAOS, TOOS, MAOS),tetrazolium salts (e.g., NTB, TV, INT, MAOS), and p-nitrophenol.

[0032] The porous membrane may be provided between the reagent layer andthe support. However, the porous membrane is merely a base which iseffective in holding a necessary portion of liquid sample for carryingout the reaction and in coating the reagent in a layer. Accordingly, itis not particularly necessary for the construction of the presentinvention. Examples of the porous membrane for use in the presentinvention include polypropylene film (e.g., Celgard produced byHoechest), polysulfone film (e.g., Filterite produced by Memtec), andpolycarbonate film (e.g., Cyclopore produced by Whatman).

[0033] In the test apparatus of the present invention, it is preferableto form a light reflection layer on the reagent layer in order tofacilitate observation of the coloring situation with the naked eye andto improve detection sensitivity by increasing the amount of light inthe detecting area. With regard to properties and size of the lightreflection layer, it is preferable to use such a thin and simple layerthat it does not react with the reagent to be used in the reagent layerand with liquid samples, does not inhibit coloration of the reagentlayer and can minimize its influence upon a permeation rate of liquidsamples as small as possible. Examples of the light reflective particlesinclude titanium dioxide, magnesium oxide, and barium sulfate. They maybe used alone, or polymer beads containing them may be used.

[0034] As described in the foregoing, unnecessary light passes throughthe light reflection layer when the light reflection layer is thinned inorder to ensure a permeation rate of liquid samples. However, it is notnecessary to take such unnecessary light into consideration in thepresent invention. In consequence, the reflection layer can be made intoa simple structure (namely a thin layer) in order to increase apermeation rate of liquid samples, and shortened measuring period can beexpected because of the apparent increase in the liquid samplepermeation rate. As an example of the reflection layer having a simplestructure, the reagent layer and light reflection layer may be made intoone body by kneading light reflective particles in the reagent layer,thereby adding light reflection function to the reagent layer itself.

[0035] The raw materials of the support and cover are not particularlylimited, provided that they do not react with reagents and liquidsamples in the reaction area and do not inhibit coloration of thereagent, or they are treated in advance for such purposes. Examples ofthe raw materials of the support include polyethylene terephthalate(referred to as “PET” hereinafter). Examples of the raw materials of thecover include plastics such as ABS resin, acrylics, polystyrene, andvinyl chloride.

[0036] With regard to the color tone of the cover, it is preferable toselect a color which substantially has no influence on the reflectedlight at a measured wavelength. The color which substantially has noinfluence on the reflected light also means a color in which lightreflection at a measured wavelength is lower or a color which fullyabsorbs a light of a measured wavelength. Specifically, the color has areflectance of less than 30%, and preferably less than 10%. When thewavelength is 405 nm, a yellow cover is preferably used.

[0037] Black is preferred because of its advantages in that it canabsorb light over a broad range of wavelengths and therefore can beapplied to simultaneous measurement of a plurality of items.

[0038] Furthermore, a transparent cover is also preferred because of itsadvantages in that it does not reflect light over a broad range ofwavelengths and therefore can be applied to simultaneous measurement ofa plurality of items.

[0039] Examples of method for preparing the cover are shown in thefollowing.

[0040] (i) A method in which the cover is directly colored.

[0041] (a) A pigment is kneaded with components (e.g., plastics) of acover composition and then molded into a cover form.

[0042] (b) A white cover is molded and then entire portion or at least apart which corresponds to the reagent layer is colored by coating,seal-application or the like.

[0043] (ii) A method in which a transparent material is used forpreparing a transparent cover.

[0044] The thickness of the layers constituting the test apparatus ofthe present invention is not particularly limited. For example,generally, the cover has a thickness of 100 to 500 μm, and the reagentlayer has a thickness of 50 to 250 μm in a wet state and 3 to 15 μm in adry state.

[0045] The present invention will now be illustrated in greater detailwith reference to Examples, but it should be understood that theinvention is not deemed to be limited thereto.

EXAMPLE 1

[0046] Test apparatus for use in the determination of blood glucose, inwhich its sample spotting side and detecting side are different fromeach other: Composition of reagent solution: Glucose oxidase 10 kuPeroxidase 20 ku 4-Aminoantipyrine 150 mgN-Ethyl-N-(2-hydroxy-3-sulfopropyl)- 200 mg 3,5-dimethylaniline 0.15 MPhosphate buffer (pH 7.0) 2 ml 4% Hydroxypropylcellulose 3 g 50 wt %Titanium dioxide solution 1 g

[0047] The test apparatus for use in Example 1 is similar to that shownin FIG. 1 and contains the structure of reagent layer and surroundingarea thereof shown in FIG. 2. Dimensions such as width and the like areshown approximately for easy understanding by expanded sectional view.

[0048] Although the reagent layer and light reflection layer are dividedwith a dotted line in FIG. 2 for easy understanding, they are actuallymade into a composite as can be understood from the aforementionedformulation, so that the reagent layer also acts as a reflection layer.

[0049] A reagent solution of the aforementioned formulation was coatedin a thickness of 100 μm on a light permeable porous film (Nuclepore) of10 μm in thickness (2 in FIG. 1) and dried at 40° C. for 1 hour, therebyobtaining a reagent layer (3 in FIG. 1) which also served as areflection layer. Thereafter, the resulting layer was cut to an area of7 mm×7 mm.

[0050] The thus prepared apparatus of porous film, with its reagentlayer being upside, was put on a support made of PET and coated with athermoplastic resin (1 in FIG. 1) having a through hole of 4 mm indiameter (11 in FIG. 1) and then adhered by thermo-compression bonding.A cover which was made of ABS resin and molded in black color(reflectance: 5.3%, 5 in FIG. 1) was further put on the resultingsupport by setting its PET side downward and adhered bythermo-compression bonding, in order to cover the reagent layer and forma sample-holding room (4 in FIG. 1) between the cover and the support,thereby obtaining a test apparatus.

[0051] Since the cover (5 in FIG. 1) has a sample-supplying hole (51 inFIG. 1) and an air exhaust hole (52 in FIG. 1), a liquid sample addeddropwise through the sample-supplying hole develops and advances by thecapillary action and gravity flow action, getting over the reagent layerand wetting it. A portion of the sample reacted with the reagents entersinto the porous film and held therein.

[0052] A 20 μl portion of each of whole blood samples having the sameglucose level but with varied hematocrit values as shown in Table 1 wasadded dropwise to the thus prepared test apparatus, and reflectance at640 nm was measured 30 seconds thereafter from the porous film sidethrough the through hole of PET using a color-difference meter. The thusobtained reflectance was converted into K/S value based on the formulaof Kubelka-Munk. Results of the measurement are shown in Table 1.

EXAMPLE 2

[0053] A test apparatus was prepared in the same manner as in Example 1,except using a transparent cover (reflectance: 5%) instead of theaforementioned black-molded ABS resin. The obtained test apparatus wasexamined in the same manner as in Example 1.

COMPARATIVE EXAMPLE 1

[0054] As a control, a test apparatus was prepared in the same manner asin Example 1, except using a cover molded in white-molded ABS resin(reflectance: 95%) instead of the aforementioned black-molded ABS resin.

[0055] In these examples, the shape of the test apparatus used herein ismerely an example and therefore is not limited thereto. TABLE 1Comparative Example 1 Example 2 Example Reflec- Reflec- Reflec-Hematocrit tance tance tance (%) (%) K/S (%) K/S (%) K/S 0 28.3 0.90826.2 1.037 36.8 0.543 25 28.4 0.903 25.9 1.059 33.9 0.644 35 28.2 0.91426.4 1.029 32.6 0.697 45 28.1 0.920 25.8 1.064 32.6 0.697 55 28.1 0.92026.2 1.038 31.9 0.727 65 28.7 0.886 27.0 0.987 33.1 0.676

[0056] When measured using the test apparatuses of the presentinvention, fluctuations between maximum and minimum values of thereflectance and K/S value were found to be 0.6% or 1.2% and 0.034 or0.77, respectively; on the other hand, such fluctuations were 4.9% and0.184 when the control test apparatus was used.

[0057] Even when the values obtained with the hematocrit values of 0%and 65% which seemed hardly to occur in reality were excluded,fluctuations between maximum and minimum values of the reflectance andK/S value were found to be 0.3% or 0.6% and 0.017 or 0.035,respectively, when the test apparatus of the present invention was used;on the other hand, such fluctuations were 2.0% and 0.083 when thecontrol test apparatus was used.

[0058] Thus, as has been described in the foregoing, since the testapparatus of the present invention is not influenced by unnecessarylight reflected into the cover passing through the reflection layer inthe reagent layer, the problem of causing fluctuation of the measuredvalues due to difference in color of respective liquid samples can beresolved.

[0059] In addition, good results can be obtained even when thereflection layer is made into a simple structure for the purpose ofincreasing a permeation rate of liquid samples, and the thus increasedpermeation rate renders possible shortening of the measuring period.

[0060] While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

What is claimed is:
 1. A test apparatus for assaying a component in aliquid sample by measuring a reflected light comprising a support havinga through hole or a light permeable area, a reagent layer having adetecting area fixed on the support to cover the through hole or lightpermeable area, and a cover which covers at least the detecting area,wherein at least a portion covering the detecting area in said cover isprocessed into a color which substantially has no influence on thereflected light at a measured wavelength.
 2. The test apparatus asclaimed in claim 1, wherein said color is black.
 3. The test apparatusas claimed in claim 1, wherein said color is transparent.
 4. The testapparatus as claimed in claim 1, wherein said color has a reflectance ofless than 30%.
 5. The test apparatus as claimed in claim 1, wherein saidcover is fixed on the support in such a manner that a sample-holdingroom is formed between the cover and the reagent layer, and said coverhas a sample-supplying hole and an air exhaust hole.
 6. The testapparatus as claimed in claim 1, wherein said cover is contacted withone side of the reagent layer opposite to the support.
 7. The testapparatus as claimed in claim 1, wherein a light reflection layer isfurther provided on the reagent layer in contact with the reagent layer.8. The test apparatus as claimed in claim 1, wherein said reagent layercontains light reflective particles.
 9. The test apparatus as claimed inclaim 1, wherein the liquid sample is whole blood.
 10. The testapparatus as claimed in claim 1, wherein a porous membrane is furtherprovided between the reagent layer and the support.